The following description provides a summary of information relevant to the present disclosure and is not a concession that any of the information provided or publications referenced herein is prior art to the claimed invention.
A key form of evidence in modern sexual assault cases is a DNA profile originating from the perpetrator. Such evidence is generally obtained from swabs taken in the course of forensic examination of victims. These swabs are typically heavily contaminated with epithelial cells from the victim as well as bearing semen from the perpetrator. The vast excess of released DNA from the victim's cells can interfere with generation of a clean DNA profile from the perpetrator, therefore a purification step is required to separate contaminating epithelia from sperm cells. Currently, the most common protocol is the so-called differential extraction (DE) procedure (Gill et al. (1985) Nature, 318(6046):577-579; Yoshida et al. (1995) Forensic Sci Int, 72(11:25-33). This method relies on differential lysis of sperm and epithelial cells in sodium dodecyl sulfate (SDS). In spite of its simplicity, it is time consuming, labor intensive, difficult to automate and can result in possible DNA mixtures when sperm head counts are low.
A variety of methods described in the literature are aimed at improving or modifying the differential extraction procedure to achieve better sperm separation from contaminating victim DNA (Voorhees et al. (2006) J Forensic Sci, 51(3):574-579; Ladd et al. (2006) NCJRS) (www.ncjrs.gov/pdffiles1/nij/grants/215339.pdf); Garvin et al. (2009) J Forensic Sci, 54(6):1297-1303). Microfluidic devices have been created which exploit the differential physical properties of sperm cells versus other cells and also allow for direct silica-based DNA extraction (Horsman et al. (2005) Anal Chem, 77(3):742-749; Bienvenue et al. (2006) J Forensic Sci, 51(2):266-273). Multiple publications describe the uses of laser microdissection to selectively capture and isolate sperm cells (Di Martino et al. (2004) Forensic Sci Int, 146 Suppl:S151-153; Elliott et al. (2003) Forensic Sci Int 137(1):28-36; Sanders et al. (2006) J Forensic Sci, 51(4):748-757). This method uses an optical microscope fitted with a laser beam to capture cells of interest from cell smears. This method offers high specificity and allows the use of limited numbers of spermatozoa on microscope slides for DNA extraction and Forensic Short Tandem Repeat (STR) analysis. However, this system is expensive; process is time-consuming, labor-intensive and not easily amenable to automation.
An automated format for the differential extraction process has been developed by Promega Corporation. The Differex system allows the processing of up to 48 samples in parallel using 96 well microtiter plates and robotic pipetting system such as Biomek 2000 from Beckman Coulter. However, this platform has not gained popularity, perhaps due to the high cost of an appropriate automation platform. Moreover, the necessity for minimization of the risk of cross-contamination and misidentification of samples essentially precludes use of an open 96-well plate. Improved methods that allow the forensic analyst to process samples individually are clearly needed.
One approach that bypasses the selective lysis and extensive cell washing by centrifugation used in differential extraction processes is to physically separate sperm cells away from intact epithelial cells. Cell sorting using flow cytometry has been demonstrated (Schoell et al. (1999) Obstet Gynecol, 94(4):623-627); Schoell et al. (1999) Cytometry, 36(4):319-323; however, it is unlikely that this technique would be widely used to casework due to cost of FACS instruments with cell sorting capacity and the difficulty in operating them. Sperm cell separation from epithelial cells can also be achieved using size filtration, for example, sperm can be physically separated from much larger epithelial cells using 10 micrometer filter (Chen et al. (1998) J Forensic Sci, 43(1):114-118, or sperm can be retained using 2 micrometer filter if epithelial cells are lysed (Ladd et al. (2006) NCJRS (www.ncjrs.gov/pdffiles1/nij/grants/215339.pdf); Garvin, A. M. (2003) J Forensic Sci, 48(5):1084-1087). These filtration methods still require centrifugation, and filters are susceptible to clogging and inefficient cell recovery. Moreover, they do not provide male DNA fractions that are as good or better that those generated by the standard differential extraction method. Laser microdissection of sperm cells from slides has been also demonstrated (Di Martino et al. (2004) Forensic Sci Int, 146 Suppl:S151-153; Elliott et al. (2003) Forensic Sci Int 137(1):28-36; Sanders et al. (2006) J Forensic Sci, 51(4):748-757). This method allows for highly specific isolation of cells, however, this method is unlikely to be widely adopted for forensic casework analysis due to the high cost of the necessary instrumentation.
Affinity purification of sperm cells is in principle one of the simplest, most intuitive, and accessible methods for processing of forensic samples. Indeed, affinity purification using antibodies to various sperm cell surface antigens has been demonstrated (Eisenberg, A. (2002) NCJRS (www.ncjrs.gov/pdffiles1/nij/grants/197532.pdf). However, this approach suffered from low efficiency, as captured cells tended to be lost in wash steps. Photocrosslinking of antibody and antigen was utilized to improve complex retention; however, this approach requires chemical modification of antibodies and has not gained popularity in actual forensic applications. Antibody epitope stability is another problem with this approach, because detergents typically used for swab elution (SDS or Sarkosyl) denature epitopes recognized by anti-sperm antibodies.